We will determine the sequence of the "gap region" on the 42S minus strand RNA in about 50% of SFV RF's and RI's which can be cleaved by low levels of pancreatic RNase to yield cores RFII and RFIII. We will copy with AMV reverse transcriptase the region into a DNA copy which we expect to be covalently linked to the 3'-terminus of the plus strand of RFII. If necessary for sequencing, we will obtain a DNA duplex using AMV reverse transcriptase or E. coli DNA polymerase I and the cDNA oligo dG as template after extension with dC residues of the 3'terminus of the cDNA with terminal transferase from calf thymus. We will attempt to restore functions which the SFV replication complex has lost during various steps in purification, functions such as initiation of RNA synthesis and release of completed RNA chains newly synthesized in vitro. The purified SFV replication complex contains three nonstructural proteins, ns70:ns86: and ns72 in a ratio of 4:1:1. Most of the replication complex sediments into a pellet at 15,000 x g, whereas from 40 to 60% of ns86 and ns72 remain in the supernatant fraction. We will purify these proteins utilizing affinity chromatography, determine their ability to synthesize RNA in response to exogenously added template; and identify what role these proteins have in the SFV replication complex.